In a flow cytometer, cells or particles tagged with markers or dyes are passed through a light beam at relatively high speed. Typically the identity of the cells or particles is determined by measuring the intensity of fluorescent emissions produced by the cells or particles or the intensity of the light scattered by the cells or particles. However, intensity measurements alone may not be adequate to identify particles. For example, the resolution of multiple signals with similar emission spectra and similar intensities, suppression of a single emitted signal from multiple signals of similar intensities, and normalization of intensity varying signals with the same or different fluorescent lifetimes are not possible using intensity measurements alone. In general, fluorescent lifetime or phase shift cannot readily be measured by intensity measurements alone.
Fluorescently tagged particles may have multiple fluorescent tags, some having similar emission spectra. Previously the only way to separate these signals with similar emissions was through differences in intensities between light emitted by cells of different types when illuminated. If the signals had similar intensities then the multiple signals could not be resolved. In contrast, the present invention allows such signals to be resolved even if they have different lifetimes and the same or different intensities.
Fluorescent lifetime is associated with the radiative transition from a fluorescent agent's excited state to its relaxed state. The excited state is dependent on the nature of the fluorescent agent. The fluorescent agent (fluorochrome) is chosen to be, or is a natural property of, the cell or particle under observation. The lifetime of the fluorescent agent may be determined by phase sensitive or modulation sensitive techniques.
The fluorescent lifetime of a cell, .tau., is dependent on the phase shift of the fluorescent emission relative to a reference signal as follows: EQU .tau.=1/.omega.(Tan.theta.)
where
.omega.=2 .pi.f, f=frequency of the excitation beam
.theta.=phase angle (shift) between the fluorescent emission and the excitation beam
The fluorescent lifetime may also be determined from the depth of modulation factor m. EQU .tau.=1/.omega.(1/m.sup.2 -1).sup.178
Fluorescent lifetime may thus be resolved through the measurement of the phase shift ".theta." between the fluorescent emission and the excitation light beam. The measured signal may include multiple lifetime signals, and therefore multiple phase signals.
While the detection of phase shift between signals is known, an apparatus and method for determination of phase fluorescence in a high speed and therefore dynamic system is not well known. Determination of the phase shift between a modulated wave and the modulated emission from the cell or particle in a flow cytometer is problematic because of the high speed at which each cell or particle passes through the flow chamber and the widely varying intensity of the signal resulting from emissions of fluorescence.
Since the phase measurement is a relative measurement, the choice of the reference from which the measurement is taken is important.
In order to obtain a reference against which the phase measurement may be taken, the intensity of the excitation light beam is modulated. An acoustic optic modulator is used to modulate the illuminating light beam. The acoustic optic modulator may be a traveling wave modulator or a standing wave modulator. It is desirable to use a traveling wave modulator rather than a standing wave modulator to modulate the illuminating light beam for several reasons including: a traveling wave modulator is cheaper than a standing wave modulator; a traveling wave modulator is able to produce a wider range of modulating frequencies than a standing wave modulator and the efficiency of a traveling wave modulator is greater than that of a standing wave modulator over a range of frequencies.
A standing wave modulator produces modulated waves, which, when viewed from a point directly in the path of the modulated light beam, have constant phase. That is to say, the beam is made up of waves whose phase is constant across any cross section through the beam which is perpendicular to the direction of propagation of the waves. In contrast, a traveling wave modulator produces a beam comprising waves whose phase is not constant across a perpendicular cross section of the beam.
A method and apparatus for detecting phase fluorescence is disclosed in copending U.S. Pat. application No. 07/705,044, now abandoned assigned to Becton, Dickinson and Company and which is incorporated herein by reference. In that application, the modulator is a standing wave modulator. The modulating frequency is provided by a frequency synthesizer which also provides the reference against which the phase fluorescence is measured.
When a traveling wave modulator is used, the use of the signal from the frequency synthesizer as a reference poses difficulties. This is because the phase of the modulated waves varies transversely across the light beam. Since the positions of particles in a flow cytometer may vary, causing them to intersect different parts of the illuminating light beam, the phase of the fluorescent light varies with the positions of the particles.